In eukaryotes, movement of material into and through the cell is mediated by the budding, transport and fusion of membrane vesicles. Some of these vesicles are surrounded by coat proteins which are thought to promote vesicle formation. Clathrin-coated vesicles are required for the uptake of important materials such as nutrients, growth hormones, cholesterol and viruses and clathrin function is required for the normal growth and development of all eukaryotic organisms. The absence of clathrin can lead to aberrant growth of even cell death. Clathrin coat are composed of clathrin triskelions and each clathrin triskelion is composed of three clathrin heavy chains (HC's) and three non-covalently associated clathrin light chains (LC's). The HC's provide the structural backbone of the clathrin lattice but the role of the LC's in clathrin function remains unclear. This research proposal will examine the function of clathrin LC's in Saccharomyces cerevisiae. There is preliminary evidence that LC's stabilize HC's. This will be investigated further by examining the HC's in cells where the clathrin LC gene has been disrupted. The region of the LC important for biological function will also be explored by examining strains that express truncated and deleted forms of the LC. Also, the regions of the HC required for interaction with the LC will be delineated using the powerful two-hybrid system. The two-hybrid system will also be used to identify proteins that interact with and possibly regulate the LC's. The identification of proteins that bind to LC's may help elucidate the proposed regulatory role of LC's. The research project will provide valuable information about the role of LC's in the function of clathrin.